Aspergillus fumigatus causes a wide spectrum of diseases including allergic syndromes, chronic pulmonary aspergillosis and acute invasive aspergillosis (IA). Triazole antifungal drugs play an important role in the management of Aspergillus diseases, as they represent first-line therapy for all forms of aspergillosis and comprise the only orally active group of antifungal agents. The rapid increase in triazole resistant Aspergillus infections is a growing public health and patient management concern. Evidence has been accumulating that A. fumigatus can develop resistance during prolonged azole treatment, which exceeds 10% in patients with chronic disease. In contrast, the resistance rates in patients with acute IA, such as with transplant patients, is rather low (<0.6%). Examining the underlying mechanism of this epidemiological dilemma has been challenging because of the low culturability of the fungus from the infection sites in patients with aspergillosis. The goal of the present proposal is to elucidate key microbial factors influencing resistance and subsequent disease progression by using a comprehensive molecular assessment of resistance associated with chronic and acute Aspergillus infections after initiation of therapy. The hypothesis being explored is that selection of drug resistance in patients with chronic disease undergoing prolonged triazole therapy has a fitness cost that renders resistant strains less pathogenic in the airways of patients, and results in less culture positivity. Bronchil alveolar lavages (BALs) and other respiratory fluids will be evaluated from a large number of patients with documented chronic and acute Aspergillus infections before and after initiation of antifungal therapy. Nucleic acid will be extracted from both culture positive and culture negative specimens. A generic Aspergillus PCR and a novel expression profiling method will be used to detect the presence of A. fumigatus; and real-time PCR assays using molecular beacon technology will be applied to recognize well-defined resistance markers in cyp51A gene, which account for >90% of the triazole resistance in A. fumigatus. A statistical correlation will be used to assess the presence of underlying resistant strains in the two populations. Most importantly, the impact of acquired triazole resistance on the pathogen fitness will be examined in vivo. The relative fitness of mutant and wild-type strains will be assessed by a mixed challenge approach in a murine model of invasive pulmonary aspergillosis. This work takes advantage of new molecular tools and strong preliminary data developed by our group, and access to important clinical respiratory specimens from patients with chronic disease, including cystic fibrosis, and acute disease. This work will greatly advance our understanding of the importance of triazole resistance in the management of patients with chronic Aspergillus and allergic disease requiring prolonged antifungal therapy.